Amelogenesis imperfecta in familial hypomagnesaemia and hypercalciuria with nephrocalcinosis caused by CLDN19 gene mutations.

BACKGROUND: Amelogenesis imperfecta (AI) is a group of genetic diseases characterised by tooth enamel defects. AI was recently described in patients with familial hypercalciuria and hypomagnesaemia with nephrocalcinosis (FHHNC) caused by CLDN16 mutations. In the kidney, claudin-16 interacts with claudin-19 to control the paracellular passage of calcium and magnesium. FHHNC can be linked to mutations in both genes. Claudin-16 was shown to be expressed during amelogenesis; however, no data are available on claudin-19. Moreover, the enamel phenotype of patients with CLDN19 mutations has never been described. In this study, we describe the clinical and genetic features of nine patients with FHHNC carrying CLDN19 mutations and the claudin-19 expression profile in rat ameloblasts. METHODS: Six FHHNC Brazilian patients were subjected to mutational analysis. Three additional French patients were recruited for orodental characterisation. The expression profile of claudin-19 was evaluated by RT-qPCR and immunofluorescence using enamel epithelium from rat incisors. RESULTS: All patients presented AI at different degrees of severity. Two new likely pathogenic variations in CLDN19 were found: p.Arg200Gln and p.Leu90Arg. RT-qPCR revealed low Cldn19 expression in ameloblasts. Confocal analysis indicated that claudin-19 was immunolocalised at the distal poles of secretory and maturing ameloblasts. CONCLUSIONS: For the first time, it was demonstrated that AI is associated with FHHNC in patients carrying CLDN19 mutations. The data suggest claudin-19 as an additional determinant in enamel formation. Indeed, the coexistence of hypoplastic and hypomineralised AI in the patients was consistent with claudin-19 expression in both secretory and maturation stages. Additional indirect systemic effects cannot be excluded.

Ameloblastin as a putative marker of specific bone compartments.

Ameloblastin (AMBN), a member of the enamel matrix protein family, has been recently identified as integral part of the skeleton beyond the enamel. However, the specific role of endogenous AMBN in bone tissue is not fully elucidated. This study aims at investigating mRNA expression of AMBN in wild-type mice in different bone sites from early embryonic to adult stages. AMBN mRNA expression started at pre-dental stages in mouse embryos (E10.5) in both head and body parts. Using laser capture microdissection on 3-day-old mice, we showed an unambiguous mRNA expression of AMBN in extra-dental tissue (mandible bone). Screening of AMBN mRNA expression in adult mice (15-week-old) revealed that mRNA expression of AMBN varied according to the bone site; a higher mRNA levels in mandibular and frontal bone compartments were observed when compared to tibia and occipital bones. These results strongly suggest that AMBN expression may be regulated in a site-specific manner and identify AMBN as a putative in vivo marker of the site-specific fingerprint of bone organs.

Enamel protein regulation and dental and periodontal physiopathology in MSX2 mutant mice.

Signaling pathways that underlie postnatal dental and periodontal physiopathology are less studied than those of early tooth development. Members of the muscle segment homeobox gene (Msx) family encode homeoproteins that show functional redundancy during development and are known to be involved in epithelial-mesenchymal interactions that lead to crown morphogenesis and ameloblast cell differentiation. This study analyzed the MSX2 protein during mouse postnatal growth as well as in the adult. The analysis focused on enamel and periodontal defects and enamel proteins in Msx2-null mutant mice. In the epithelial lifecycle, the levels of MSX2 expression and enamel protein secretion were inversely related. Msx2+/- mice showed increased amelogenin expression, enamel thickness, and rod size. Msx2-/- mice displayed compound phenotypic characteristics of enamel defects, related to both enamel-specific gene mutations (amelogenin and enamelin) in isolated amelogenesis imperfecta, and cell-cell junction elements (laminin 5 and cytokeratin 5) in other syndromes. These effects were also related to ameloblast disappearance, which differed between incisors and molars. In Msx2-/- roots, Malassez cells formed giant islands that overexpressed amelogenin and ameloblastin that grew over months. Aberrant expression of enamel proteins is proposed to underlie the regional osteopetrosis and hyperproduction of cellular cementum. These enamel and periodontal phenotypes of Msx2 mutants constitute the first case report of structural and signaling defects associated with enamel protein overexpression in a postnatal context.

Experimental periodontitis in Msx2 mutant mice induces alveolar bone necrosis.

BACKGROUND: Msx2 homeoprotein is a key transcription factor of dental and periodontal tissue formation and is involved in many molecular pathways controlling mineralized tissue homeostasis such as Wnt/sclerostin pathway. This study evaluated the effect of Msx2-null mutation during experimental periodontitis in mice. METHODS: Experimental periodontitis was induced for 30 days in wild-type and Msx2 knock-in Swiss mice using Porphyromonas gingivalis infected ligatures. In knock-in mice, Msx2 gene was replaced by n-LacZ gene encoding ß-galactosidase. Periodontal tissue response was assessed by histomorphometry, tartrate-resistant acid phosphatase histoenzymology, ß-galactosidase, sclerostin immunochemistry, and terminal deoxynucleotidyl transferase-mediated dUTP nickend labeling assay. Expression of Msx2 gene expression was also evaluated in human gingival biopsies using RT-qPCR. RESULTS: During experimental periodontitis, osteonecrosis area and osteoclast number were significantly elevated in knock-in mice compared with wild-type mice. Epithelial downgrowth and bone loss was similar. Sclerostin expression in osteocytes appeared to be reduced during periodontitis in knock-in mice. Msx2 expression was detected in healthy and inflamed human gingival tissues. CONCLUSION: These data indicated that Msx2 pathway influenced periodontal tissue response to experimental periodontitis and appeared to be a protective factor against alveolar bone osteonecrosis. As shown in other inflammatory processes such as atherothrombosis, genes initially characterized in early development could also play an important role in human periodontal pathogenesis.

Autoregulatory loop of Msx1 expression involving its antisense transcripts.

The Msx1 homeogene plays an important role in epithelial-mesenchymal interactions leading organogenesis. Msx1 gene is submitted to bidirectional transcription generating a long non-coding antisense (AS) RNA potentially involved in Msx1 expression regulation. RT-Q-PCR and RNA-FISH studies indicated that transient overexpression of the Msx1 AS transcript in 705IC5 mouse odontoblasts decreased the abundance of endogenous Msx1 S mRNA at the post-transcriptional level. Conversely, Msx1 overexpression increased the AS RNA level probably by activating AS transcription. In vivo mapping by RT-PCR evidenced both Msx1 RNAs in all adult mouse tissues tested raising the issue of Msx1 function during adulthood. The expression patterns of the two RNAs were similar, confirming the tight S/AS relationship. In particular, both Msx1 mRNAs and Msx1 protein were similarly distributed in eyes, and were found in regions with a common ectodermic origin and in cells potentially involved in regeneration. In conclusion, we report that Msx1 S RNA is negatively controlled by its AS RNA at a post-transcriptional level, and that the AS RNA is retrocontrolled positively by Msx1. The tight link between Msx1 S and AS RNAs constitutes a regulatory loop resulting in a fine-tuned expression of Msx1 which appears to be significant for adult homeostasis.

Msx and dlx homeogene expression in epithelial odontogenic tumors.

Epithelial odontogenic tumors are rare jaw pathologies that raise clinical diagnosis and prognosis dilemmas notably between ameloblastomas and clear cell odontogenic carcinomas (CCOCs). In line with previous studies, the molecular determinants of tooth development-amelogenin, Msx1, Msx2, Dlx2, Dlx3, Bmp2, and Bmp4-were analyzed by RT-PCR, ISH, and immunolabeling in 12 recurrent ameloblastomas and in one case of CCOC. Although Msx1 expression imitates normal cell differentiation in these tumors, other genes showed a distinct pattern depending on the type of tumor and the tissue involved. In benign ameloblastomas, ISH localized Dlx3 transcripts and inconstantly detected Msx2 transcripts in epithelial cells. In the CCOC, ISH established a lack of both Dlx3 and Msx2 transcripts but allowed identification of the antisense transcript of Msx1, which imitates the same scheme of distribution between mesenchyme and epithelium as in the cup stage of tooth development. Furthermore, while exploring the expression pattern of signal molecules by RT-PCR, Bmp2 was shown to be completely inactivated in the CCOC and irregularly noticeable in ameloblastomas. Bmp4 was always expressed in all the tumors. Based on the established roles of Msx and Dlx transcription factors in dental cell fates, these data suggest that their altered expression is a proposed trail to explain the genesis and/or the progression of odontogenic tumors.

Differential impact of MSX1 and MSX2 homeogenes on mouse maxillofacial skeleton.

Craniofacial development involves a large number of genes involved in a complex time- and site-specific cascade of cellular crosstalk. Msx homeobox genes are expressed very early and have been implicated in multiple signaling processes. However, little is known about their role in postnatal growth and at adult stages. The aim of this study was to compare the patterns of expression of Msx1 and Msx2 during postnatal growth and homeostasis. We used transgenic mice with a knock-in for Msx1 or Msx2. Msx expression was analyzed on whole-mount experiments on heterozygous mice. The results were confirmed by quantitative RT-PCR on mandible and tibia samples. Steady-state levels of Msx2 mRNA were determined at 2 ages, at postnatal day 14 and after 3 months, corresponding to phases of growth and homeostasis, respectively. Consistent with previous findings, the expression profiles of Msx1 and Msx2 overlapped during embryonic development. By contrast, marked differences in the patterns of expression of these 2 genes were observed during the growth phase. Msx1 was found to be expressed in basal bone during postnatal growth. Msx1 was not expressed in alveolar bone, whereas Msx2 was strongly and continually expressed. Msx2 was present in all growth plate cartilages, as previously shown for Msx1. Autopods displayed different patterns of expression during the mouse life cycle, with continuous expression of Msx1 only. Interestingly, both secretory cells (osteoblasts) and cells involved in bone resorption (osteoclasts) were found to be involved in Msx molecular pathways, their precise involvement depending on the anatomical site. The observed patterns correspond to specific sites during growth and constitute landmarks in our understanding of growth-related oral facial dysmorphologies.

Physiological implications of DLX homeoproteins in enamel formation.

Tooth development is a complex process including successive stages of initiation, morphogenesis, and histogenesis. The role of the Dlx family of homeobox genes during the early stages of tooth development has been widely analyzed, while little data has been reported on their role in dental histogenesis. The expression pattern of Dlx2 has been described in the mouse incisor; an inverse linear relationship exists between the level of Dlx2 expression and enamel thickness, suggesting a role for Dlx2 in regulation of ameloblast differentiation and activity. In vitro data have revealed that DLX homeoproteins are able to regulate the expression of matrix proteins such as osteocalcin. The aim of the present study was to analyze the expression and function of Dlx genes during amelogenesis. Analysis of Dlx2/LacZ transgenic reporter mice, Dlx2 and Dlx1/Dlx2 null mutant mice, identified spatial variations in Dlx2 expression within molar tooth germs and suggests a role for Dlx2 in the organization of preameloblastic cells as a palisade in the labial region of molars. Later, during the secretory and maturation stages of amelogenesis, the expression pattern in molars was found to be similar to that described in incisors. The expression patterns of the other Dlx genes were examined in incisors and compared to Dlx2. Within the ameloblasts Dlx3 and Dlx6 are expressed constantly throughout presecretory, secretory, and maturation stages; during the secretory phase when Dlx2 is transitorily switched off, Dlx1 expression is upregulated. These data suggest a role for DLX homeoproteins in the morphological control of enamel. Sequence analysis of the amelogenin gene promoter revealed five potential responsive elements for DLX proteins that are shown to be functional for DLX2. Regulation of amelogenin in ameloblasts may be one method by which DLX homeoproteins may control enamel formation. To conclude, this study establishes supplementary functions of Dlx family members during tooth development: the participation in establishment of dental epithelial functional organization and the control of enamel morphogenesis via regulation of amelogenin expression.

Expression pattern of Dlx3 during cell differentiation in mineralized tissues.

The present study was designed to compare the expression pattern of Dlx3 in four different mineralized tissues because of: 1-its role in skeleton patterning, 2-its expression in dental epithelium and mesenchyme during morphogenesis, 3-the membranous and endochondral bone and tooth phenotype of tricho-dento-osseous syndrome related to Dlx3 gene mutation and 4-recently emerging knowledge on Dlx family members in the bone field. Ameloblasts, odontoblasts, osteoblasts and chondrocytes were analyzed in vitro and in vivo. Dlx3 transcripts were detected by RT-PCR in established model systems (microdissected dental epithelium and mesenchyme; primary cultures of rat chondrocytes), as recently performed in osteoblasts in vitro. A human 414-bp Dlx3 probe was generated. A 4.5-kb human Dlx3 sense RNA was identified in maxillo-facial samples by Northern blotting. Immunolabeling and in situ hybridization were performed in mice from Theiler stage E 14.5 until birth. In teeth, although Dlx3 was still expressed in differentiated ameloblasts, it was down regulated during odontoblast polarization. During endochondral bone formation, Dlx3 protein was detected in chondrocytes and was most strongly expressed in the prehypertrophic cartilage zone and in differentiating and differentiated osteoblasts of metaphyseal periosteum. In vitro, real-time PCR studies supported this upregulation in prehypertrophic chondrocytes, closely correlated with Ihh variations. In membranous bone, Dlx3 was present in preosteoblasts, osteoblasts and osteoid-osteocytes. The present data on Dlx3 and recently published functional studies show that this transcription factor may be instrumental during growth in the control of matrix deposition and biomineralization in the entire skeleton.

Msx1 role in craniofacial bone morphogenesis.

The homeobox gene Msx1 encodes a transcription factor that is highly expressed during embryogenesis and postnatal development in bone. Mutations of the MSX1 gene in humans are associated with cleft palate and (or) tooth agenesis. A similar phenotype is observed in newborn mice invalidated for the Msx1 gene. However, little is known about Msx1 function in osteoblast differentiation and bone mineralization in vivo. In the present study, we aimed to explore the variations of individualized bone shape in a subtle way avoiding the often severe consequences associated with gene mutations. We established transgenic mice that specifically express Msx1 in mineral-matrix-secreting cells under the control of the mouse 2.3kb collagen 1 alpha 1 (Col1α1) promoter, which enabled us to investigate Msx1 function in bone in vivo. Adult transgenic mice (Msx1-Tg) presented altered skull shape and mineralization resulting from increased Msx1 expression during bone development. Serial section analysis of the mandibles showed a high amount of bone matrix in these mice. In addition, osteoblast number, cell proliferation and apoptosis were higher in Msx1-Tg mice than in controls with regional differences that could account for alterations of bone shape. However, Von Kossa staining and µCT analysis showed that bone mineralization was lower in Msx1-Tg mice than in controls due to alteration of osteoblastic differentiation. Msx1 appears to act as a modeling factor for membranous bone; it stimulates trabecular bone metabolism but limits cortical bone growth by promoting apoptosis, and concomitantly controls the collagen-based mineralization process.

Tracking endogenous amelogenin and ameloblastin in vivo.

Research on enamel matrix proteins (EMPs) is centered on understanding their role in enamel biomineralization and their bioactivity for tissue engineering. While therapeutic application of EMPs has been widely documented, their expression and biological function in non-enamel tissues is unclear. Our first aim was to screen for amelogenin (AMELX) and ameloblastin (AMBN) gene expression in mandibular bones and soft tissues isolated from adult mice (15 weeks old). Using RT-PCR, we showed mRNA expression of AMELX and AMBN in mandibular alveolar and basal bones and, at low levels, in several soft tissues; eyes and ovaries were RNA-positive for AMELX and eyes, tongues and testicles for AMBN. Moreover, in mandibular tissues AMELX and AMBN mRNA levels varied according to two parameters: 1) ontogenic stage (decreasing with age), and 2) tissue-type (e.g. higher level in dental epithelial cells and alveolar bone when compared to basal bone and dental mesenchymal cells in 1 week old mice). In situ hybridization and immunohistodetection were performed in mandibular tissues using AMELX KO mice as controls. We identified AMELX-producing (RNA-positive) cells lining the adjacent alveolar bone and AMBN and AMELX proteins in the microenvironment surrounding EMPs-producing cells. Western blotting of proteins extracted by non-dissociative means revealed that AMELX and AMBN are not exclusive to mineralized matrix; they are present to some degree in a solubilized state in mandibular bone and presumably have some capacity to diffuse. Our data support the notion that AMELX and AMBN may function as growth factor-like molecules solubilized in the aqueous microenvironment. In jaws, they might play some role in bone physiology through autocrine/paracrine pathways, particularly during development and stress-induced remodeling.

Regulation of calbindin-D(28k) expression by Msx2 in the dental epithelium.

Amelogenesis involves the coordinated expression of a set of molecules that includes enamel matrix proteins and calcium-binding proteins. Msx2 is a member of the divergent homeobox gene family and is instrumental in dental morphogenesis and biomineralization. This study focused on an EF-hand calcium-binding protein, calbindin-D(28k), which is highly expressed in dental epithelium. In vivo data showed that calbindin-D(28k) levels were higher in ameloblasts from Msx2(+/-) mice than Msx2(+/+) mice. Consistent with this finding, calbindin-D(28k) distribution was affected in transgenic mice with ectopic expression in root epithelium in rests of Malassez in Msx2(+/-) and more clearly in Msx2(-/-) mice. In accordance with these in vivo data, calbindin-D(28k) protein and mRNA levels were decreased in LS8 ameloblast-like cells by exogenous Msx2 overexpression. Furthermore, calbindin-D(28k) promoter activity (nt-1075/+34) was specifically diminished in the presence of Msx2 overexpression, showing that Msx2 behave as a transcriptional repressor for calbindin-D(28k) gene expression. In conclusion, Msx2 may control the spatiotemporally restricted frame of calbindin-D(28k) production in the dental epithelium in relation to enamel mineralization, as previously shown for amelogenin.

Vitamin D and tissue non-specific alkaline phosphatase in dental cells.

Dental epithelium comprises different cell populations, including ameloblasts and stratum intermedium cells. Ameloblasts are vitamin D targets, and at least five proteins undergo specific modulation of their expression following the addition of 1alpha,25(OH)2 vitamin D3[1alpha,25(OH)2D3]. Stratum intermedium cells have not been studied in any great detail regarding vitamin D impact. Interestingly, in these cells, the tissue non-specific alkaline phosphatase (TNAP) is overexpressed. On the other hand, TNAP is a reliable bone marker of vitamin D action, similar to calbindins in kidney and intestine, previously used for studies of vitamin D activity in ameloblasts. Here, TNAP expression and activity were investigated in vivo in the microdissected epithelium and mesenchyme of mandible incisors. Physiological doses of 1alpha,25(OH)2D3 injected in control rats failed to modify TNAP activity in both dental epithelium and mesenchyme. No significant differences were observed in the steady-state levels of TNAP mRNAs of dental tissues from wild-type and vitamin D nuclear receptor (VDRnuc)-deficient mice of the same litters. These data suggest that, in contrast to ameloblasts, stratum intermedium cells are not sensitive to 1alpha,25(OH)2D3. An explanation for such a responsiveness of stratum intermedium cells to 1alpha,25(OH)2D3 is proposed based on the respective expressions of both vitamin D receptors (VDRnuc and 1,25D3-[MARRS]) and the Dlx2 homeobox gene.

Modulation of 1alpha,25-dihydroxyvitamin D3-membrane associated, rapid response steroid binding protein expression in mouse odontoblasts by 1alpha,25-(OH)2D3.

The rapid, nongenomic effects of 1alpha,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3 have been related to a 1,25D3-membrane associated, rapid response steroid binding protein or 1,25D3-[MARRS]bp, with a molecular weight of 65 kDa, in several tissues and species. Currently, no information is available concerning the nongenomic responses to 1alpha,25-(OH)2D3 in dental tissues. In order to investigate the expression of 1,25D3-[MARRS]bp in dental cells, in the presence or absence of 1alpha,25-(OH)2D3, we have used rabbit polyclonal antibodies directed against the N-terminus of the 1,25D3-[MARRS]bp (Ab099) that recognizes the 1alpha,25-(OH)2D3 binding protein in chick intestinal basolateral membranes and a mouse odontoblast-like cell line (MO6-G3). Western blotting and flow cytometric analyses with Ab099 specifically detected 1,25D3-[MARRS]bp in MO6-G3 cells. Moreover, 1,25D3-[MARRS]bp was up-regulated, in vivo, in differentiated dental cells. Electron microscopic analysis confirmed the plasma membrane localization of this binding protein and also showed its intracellular presence. Incubation of MO6-G3 cells with different doses of 1alpha,25-(OH)2D3 for 36 h resulted in an inhibition of 1,25D3-[MARRS]bp expression with a maximal effect at 50 nM steroid. In addition, the culture media of MO6-G3 cells contains immunoreactive 1,25D3-[MARRS]bp. Immunogold positive membrane vesicle-like structures are present in the extracellular matrix of MO6-G3 cells. Altogether, these results indicate that the 1,25D3-[MARRS]bp expression in MO6-G3 cells is modulated by 1alpha,25-(OH)2D3. In conclusion, this 1alpha,25-(OH)2D3 binding protein could play an important role in the rapid, nongenomic responses to 1alpha,25-(OH)2D3 in dental cells.